Gene silencing in mammalian cells through RNA interference (RNAi) has become an invaluable tool for the study of biological processes. RNAi is mediated through the action of small 21 nucleotide duplex RNAs, known as small interfering RNAs (siRNAs), which form part of an endogenous enzyme complex, termed the RNA-induced silencing complex (RISC). More specifically, a single strand of the siRNA duplex is selectively loaded into RISC where it provides stringent guidance for the catalytic cleavage of complementary mRNA transcripts. We are employing RNAi for the exploration of cancer-associated pathways including the validation of anti-cancer targets, the potential identification of new targets, the development of novel model systems, and the elucidation/validation of integral components of cancer related phenotypes. To access large-scale synthetic siRNA resources GSS has developed a research collaboration agreement with Qiagen Inc. To date Qiagen has designed and synthesized siRNAs corresponding to approximately 400 genes. To make full use of these resources have developed a high throughput automated synthetic siRNA-lipid "reverse transfection" protocol in a 96 well plate format (BioRobot 8000, Qiagen Inc.). To date we have assayed the knockdown mediated by 267 siRNAs corresponding to 131 human genes. Over 70% of these siRNAs show a statistically significant decrease in the steady state levels of the expression of the gene under study. We are actively investigating the reasons for why some siRNAs fail to silence. In many cases we suspect that a failure to induce silencing often reflects issues related to the expression of different alternative transcripts of the gene in question and single nucleotide polymorphisms that block cleavage by the siRNA-RISC complex. In a limited number of cases we have also investigated the specificity of the siRNAs. In addition to validation we are now expanding this platform to allow us to further our pharmocogenomic studies through the development of a combinatorial library approach.